Gene sms 27

ABSTRACT

The present invention relates to newly identified genes that encode proteins that are involved in the synthesis of L-ascorbic acid (hereinafter also referred to as Vitamin C). The invention also features polynucleotides comprising the full-length polynucleotide sequences of the novel genes and fragments thereof, the novel polypeptides encoded by the polynucleotides and fragments thereof, as well as their functional equivalents. The present invention also relates to the use of said polynucleotides and polypeptides as biotechnological tools in the production of Vitamin C from microorganisms, whereby a modification of said polynucleotides and/or encoded polypeptides has a direct or indirect impact on yield, production, and/or efficiency of production of the fermentation product in said microorganism. Also included are methods/processes of using the polynucleotides and modified polynucleotide sequences to transform host microorganisms. The invention also relates to genetically engineered microorganisms and their use for the direct production of Vitamin C.

The present invention relates to newly identified genes that encodeproteins that are involved in the synthesis of L-ascorbic acid(hereinafter also referred to as Vitamin C) and/or 2-keto-L-gulonic acid(hereinafter also referred to as 2-KGA). The invention also featurespolynucleotides comprising the full-length polynucleotide sequences ofthe novel genes and fragments thereof, the novel polypeptides encoded bythe polynucleotides and fragments thereof, as well as their functionalequivalents. The present invention also relates to the use of saidpolynucleotides and polypeptides as biotechnological tools in theproduction of Vitamin C and/or 2-KGA from microorganisms, whereby amodification of said polynucleotides and/or encoded polypeptides has adirect or indirect impact on yield, production, and/or efficiency ofproduction of the fermentation product in said microorganism. Alsoincluded are methods/processes of using the polynucleotides and modifiedpolynucleotide sequences to transform host microorganisms. The inventionalso relates to genetically engineered microorganisms and their use forthe direct production of Vitamin C and/or 2-KGA.

Vitamin C is one of very important and indispensable nutrient factorsfor human beings. Vitamin C is also used in animal feed even though somefarm animals can synthesize it in their own body.

For the past 70 years, Vitamin C has been produced industrially fromD-glucose by the well-known Reichstein method. All steps in this processare chemical except for one (the conversion of D-sorbitol to L-sorbose),which is carried out by microbial conversion. Since its initialimplementation for industrial production of Vitamin C, several chemicaland technical modifications have been used to improve the efficiency ofthe Reichstein method. Recent developments of Vitamin C production aresummarized in Ullmann's Encyclopedia of Industrial Chemistry, 5^(th)Edition, Vol. A27 (1996), pp. 547ff.

Different intermediate steps of Vitamin C production have been performedwith the help of microorganisms or enzymes isolated therefrom. Thus,2-keto-L-gulonic acid (2-KGA), an intermediate compound that can bechemically converted into Vitamin C by means of an alkalinerearrangement reaction, may be produced by a fermentation processstarting from L-sorbose or D-sorbitol, by means of strains belonginge.g. to the Ketogulonicigenium or Gluconobacter genera, or by analternative fermentation process starting from D-glucose, by means ofrecombinant strains belonging to the Gluconobacter or Pantoea genera.

Current chemical production methods for Vitamin C have some undesirablecharacteristics such as high-energy consumption and use of largequantities of organic and inorganic solvents. Therefore, over the pastdecades, other approaches to manufacture Vitamin C using microbialconversions, which would be more economical as well as ecological, havebeen investigated.

Direct Vitamin C production from a number of substrates includingD-sorbitol, L-sorbose and L-sorbosone has been reported in severalmicroorganisms, such as algae, yeast and acetic acid bacteria, usingdifferent cultivation methods. Examples of known bacteria able todirectly produce Vitamin C include, for instance, strains from thegenera of Gluconobacter, Gluconacetobacter, Acetobacter,Ketogulonicigenium, Pantoea, Pseudomonas or Escherichia. Examples ofknown yeast or algae include, e.g., Candida, Saccharomyces,Zygosaccharomyces, Schizosaccharomyces, Kluyveromyces or Chlorella.

Microorganisms able to assimilate D-sorbitol for growth usually possessenzymes able to oxidize this compound into a universal assimilationsubstrate such as D-fructose. Also microorganisms able to grow onL-sorbose possess an enzyme, NAD(P)H-dependent L-sorbose reductase,which is able to reduce this compound to D-sorbitol, which is thenfurther oxidized into D-fructose. D-fructose is an excellent substratefor the growth of many microorganisms, after it has been phosphorylatedby means of a D-fructose kinase.

For instance, in the case of acetic acid bacteria, which are obligateaerobe, gram-negative microorganisms belonging to the genus Acetobacter,Gluconobacter, and Gluconacetobacter, these microorganisms are able totransport D-sorbitol into the cytosol and convert it into D-fructose bymeans of a cytosolic NAD-dependent D-sorbitol dehydrogenase. Someindividual strains, such as Gluconobacter oxydans IFO 3292, and IFO3293, are able as well to transport L-sorbose into the cytosol andreduce it to D-sorbitol by means of a cytosolic NAD(P)H-dependentL-sorbose reductase, which then is further oxidized into D-fructose. Inthese bacteria, the Embden-Meyerhof-Parnas pathway, as well as thetricarboxyclic acid cycle are not fully active, and the main pathwaychanneling sugars into the central metabolism is the pentose phosphatepathway. D-fructose-6-phosphate, obtained from D-fructose by aphosphorylation reaction enters the pentose phosphate pathway, beingfurther metabolized and producing reducing power in the form of NAD(P)Hand tricarboxylic compounds necessary for growth and maintenance.

Acetic acid bacteria are well known for their ability to incompletelyoxidize different substrates such as alcohols, sugars, sugar alcoholsand aldehydes. These processes are generally known as oxidativefermentations or incomplete oxidations, and they have been wellestablished for a long time in the food and chemical industry,especially in vinegar and in L-sorbose production. A useful productknown to be obtained from incomplete oxidations of D-sorbitol orL-sorbose using strains belonging to the Gluconobacter genus is 2-KGA.

Acetic acid bacteria accomplish these incomplete oxidation reactions bymeans of different dehydrogenases located either in the periplasmicspace, on the periplasmic membrane as well as in the cytoplasm.Different co-factors are employed by the different dehydrogenases, themost common being PQQ and FAD for membrane-bound or periplasmic enzymes,and NAD/NADP for cytoplasmic enzymes.

While all products of these oxidation reactions diffuse back to theexternal aqueous environment through the outer membrane, some of themcan be passively or actively transported into the cell and be furtherused in metabolic pathways responsible for growth and energy formation.Inside the cell, oxidized products can many times be reduced back totheir original substrate by means of reductases, and then be channeledback to the central metabolism.

Proteins, in particular enzymes and transporters, that are active in themetabolization of D-sorbitol or L-sorbose are herein referred to asbeing involved in the Sorbitol/Sorbose Metabolization System. Suchproteins are abbreviated herein as SMS proteins and function in thedirect metabolization of D-sorbitol or L-sorbose. Metabolization ofD-sorbitol or L-sorbose includes on one side the assimilation of thesecompounds into the cytosol and further conversion into metabolitesuseful for assimilation pathways such as the Embden-Meyerhof-Parnaspathway, the pentose phosphate pathway, the Entner-Doudoroff pathway,and the tricarboxyclic acid cycle, all of them involved in all vitalenergy-forming and anabolic reactions necessary for growth andmaintenance of living cells. On the other side, metabolization ofD-sorbitol or L-sorbose also includes the conversion of these compoundsinto further oxidized products such as L-sorbosone, 2-KGA and Vitamin Cby so-called incomplete oxidation processes.

An object of the present invention is to improve the yields and/orproductivity of Vitamin C and/or 2-KGA production.

Surprisingly, it has now been found that SMS proteins or subunits ofsuch proteins having activity towards or which are involved in theassimilation or conversion of D-sorbitol, L-sorbose or L-sorbosone playan important role in the biotechnological production of Vitamin C and/or2-KGA.

In one embodiment, SMS proteins of the present invention are selectedfrom oxidoreductases [EC 1], preferably oxidoreductases acting on theCH—OH group of donors [EC 1.1], more preferably oxidoreductases withNAD⁺ or NADP⁺ as acceptor [EC 1.1.1] and oxidoreductases with otheracceptors [EC 1.1.99], most preferably selected from oxidoreductasesbelonging to enzyme classes [EC 1.1.1.1], [EC 1.1.1.15] or [EC 1.2.1.−],or preferably oxidoreductases acting on the aldehyde or oxo group ofdonors [EC 1.2], more preferably oxidoreductases with NAD⁺ or NADP⁺ asacceptor [EC 1.2.1].

Furthermore, the SMS proteins of the present invention may be selectedfrom the group consisting of membrane-bound PQQ-dependent D-sorbitoldehydrogenase, membrane-bound L-sorbose dehydrogenase, membrane-boundL-sorbosone dehydrogenase, membrane-bound FAD-dependent D-sorbitoldehydrogenase, cytosolic NAD-dependent D-sorbitol dehydrogenase,NAD(P)-dependent D-sorbitol dehydrogenase (also called asNADPH-dependent sorbose reductase), NAD-dependent xylitol dehydrogenase,NAD-dependent alcohol dehydrogenase, membrane-bound L-sorbosedehydrogenase, NAD(P)H-dependent L-sorbose reductase, cytosolicNADP-dependent sorbosone dehydrogenase, cytosolic NAD(P)H-dependentL-sorbosone reductase, membrane-bound aldehyde dehydrogenase, cytosolicaldehyde dehydrogenase, glycerol-3-phosphate dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase, and others involved in SMSincluding proteins involved in the regulation of the genes encodingthese enzymes, such as e.g. transcription factors, in particularrepressors.

In particular, it has now been found that SMS proteins encoded bypolynucleotides having a nucleotide sequence that hybridizes preferablyunder highly stringent conditions to a sequence shown in SEQ ID NO:1play an important role in the biotechnological production of Vitamin Cand/or 2-KGA. It has also been found, that by genetically altering theexpression level of nucleotides according to the invention in amicroorganism capable of directly producing Vitamin C, such as forexample Gluconobacter, the direct fermentation of Vitamin C and/or 2-KGAby said microorganism can be even greatly improved.

Consequently, the invention relates to a polynucleotide selected fromthe group consisting of:

(a) polynucleotides encoding a polypeptide comprising the amino acidsequence according to SEQ ID NO:2;(b) polynucleotides comprising the nucleotide sequence according to SEQID NO:1;(c) polynucleotides comprising a nucleotide sequence obtainable bynucleic acid amplification such as polymerase chain reaction, usinggenomic DNA from a microorganism as a template and a primer setaccording to SEQ ID NO:3 and SEQ ID NO:4;(d) polynucleotides comprising a nucleotide sequence encoding a fragmentor derivative of a polypeptide encoded by a polynucleotide of any of (a)to (c) wherein in said derivative one or more amino acid residues areconservatively substituted compared to said polypeptide, and saidfragment or derivative has the activity of a transcriptional regulator,preferably a repressor of L-sorbose dehydrogenase (SDH) and/orL-sorbosone dehydrogenase (SNDH) (SMS 27);(e) polynucleotides the complementary strand of which hybridizes understringent conditions to a polynucleotide as defined in any one of (a) to(d) and which encode a transcriptional regulator, preferably a repressorof L-sorbose dehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH)(SMS 27); and(f) polynucleotides which are at least 60%, such as 70, 85, 90 or 95%identical to a polynucleotide as defined in any one of (a) to (d) andwhich encode a transcriptional regulator, preferably a repressor ofL-sorbose dehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH)(SMS 27); orthe complementary strand of such a polynucleotide.

The SMS protein as isolated from Gluconobacter oxydans IFO 3293 shown inSEQ ID NO:2 and described herein was found to be a particularly usefulSMS protein, since it appeared that it performs a crucial function inthe direct Vitamin C production in microorganisms, in particular inbacteria, such as acetic acid bacteria, such as Gluconobacter,Acetobacter and Gluconacetobacter. Accordingly, the invention relates toa polynucleotide encoding a polypeptide according to SEQ ID NO:2. Thisprotein may be encoded by a nucleotide sequence as shown in SEQ ID NO:1.The invention therefore also relates to polynucleotides comprising thenucleotide sequence according to SEQ ID NO:1.

The nucleotide and amino acid sequences determined above were used as a“query sequence” to perform a search with Blast2 program (version 2 orBLAST from National Center for Biotechnology [NCBI] against the databasePRO SW-SwissProt (full release plus incremental updates). From thesearches, the SMS 27 polynucleotide according to SEQ ID NO:1 wasannotated as encoding a transcriptional regulator belonging to theTetR/AcrR-family. The protein as encoded by SEQ ID NO:2 acts as arepressor by directly binding to the promoter region of the respectivegenes, including the genes coding for L-sorbose dehydrogenase (SDH),such as e.g. shown in SEQ ID NO:11 encoding a protein as of SEQ IDNO:12, L-sorbosone dehydrogenase (SNDH), such as e.g. shown in SEQ IDNO:13 encoding a protein as of SEQ ID NO:14, and L-sorbosone exporter,such as e.g. shown in SEQ ID NO:15 encoding a protein as of SEQ IDNO:16.

A nucleic acid according to the invention may be obtained by nucleicacid amplification using cDNA, mRNA or alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers such as the nucleotideprimers according to SEQ ID NO:3 and SEQ ID NO:4 according to standardPCR amplification techniques. The nucleic acid thus amplified may becloned into an appropriate vector and characterized by DNA sequenceanalysis.

The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from strains known or suspected tocomprise a polynucleotide according to the invention. The PCR productmay be subcloned and sequenced to ensure that the amplified sequencesrepresent the sequences of a new nucleic acid sequence as describedherein, or a functional equivalent thereof.

The PCR fragment may then be used to isolate a full length cDNA clone bya variety of known methods. For example, the amplified fragment may belabeled and used to screen a bacteriophage or cosmid cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary.

Accordingly, the invention relates to polynucleotides comprising anucleotide sequence obtainable by nucleic acid amplification such aspolymerase chain reaction, using DNA such as genomic DNA from amicroorganism as a template and a primer set according to SEQ ID NO:3and SEQ ID NO:4.

The invention also relates to polynucleotides comprising a nucleotidesequence encoding a fragment or derivative of a polypeptide encoded by apolynucleotide as described herein wherein in said derivative one ormore amino acid residues are conservatively substituted compared to saidpolypeptide, and said fragment or derivative has the activity of a SMSpolypeptide, preferably a SMS 27 polypeptide.

The invention also relates to polynucleotides the complementary strandof which hybridizes under stringent conditions to a polynucleotide asdefined herein and which encode a SMS polypeptide, preferably a SMS 27polypeptide.

The invention also relates to polynucleotides which are at least 60%identical to a polynucleotide as defined herein and which encode a SMSpolypeptide; and the invention also relates to polynucleotides being thecomplementary strand of a polynucleotide as defined herein above.

The invention also relates to primers, probes and fragments that may beused to amplify or detect a DNA according to the invention and toidentify related species or families of microorganisms also carryingsuch genes.

The present invention also relates to vectors which includepolynucleotides of the invention and microorganisms which aregenetically engineered with the polynucleotides or said vectors.

The invention also relates to processes for producing microorganismscapable of expressing a polypeptide encoded by the above definedpolynucleotide and a polypeptide encoded by a polynucleotide as definedabove.

The invention also relates to microorganisms wherein the activity of aSMS polypeptide, preferably a SMS 27 polypeptide, is reduced orabolished so that the yield of Vitamin C and/or 2-KGA which is directlyproduced from D-sorbitol or L-sorbose is increased.

The skilled person will know how to reduce or abolish the activity of aSMS protein, preferably a SMS 27 protein. Such may be for instanceaccomplished by either genetically modifying the host organism in such away that it produces less or no copies of the SMS protein, preferablythe SMS 27 protein, than the wild type organism or by decreasing orabolishing the specific activity of the SMS protein, preferably the SMS27 protein.

In the following description, procedures are detailed to achieve thisgoal, i.e. the increase in the yield and/or production of Vitamin Cwhich is directly produced from D-sorbitol or L-sorbose by reducing orabolishing the activity of a SMS 27 protein. These procedures applymutatis mutandis for other SMS proteins.

Modifications in order to have the organism produce less or no copies ofthe SMS 27 gene and/or protein may include the use of a weak promoter,or the mutation (e.g. insertion, deletion or point mutation) of (partsof) the SMS 27 gene or its regulatory elements. Decreasing or abolishingthe specific activity of a SMS 27 protein may also be accomplished bymethods known in the art. Such methods may include the mutation (e.g.insertion, deletion or point mutation) of (parts of) the SMS 27 gene.This may for instance affect the interaction with DNA that is mediatedby the N-terminal region of SMS 27 or interaction with other effectormolecules.

Also known in the art are methods of reducing or abolishing the activityof a given protein by contacting the SMS 27 protein with specificinhibitors or other substances that specifically interact with the SMS27 protein. In order to identify such specific inhibitors, the SMS 27protein may be expressed and tested for activity in the presence ofcompounds suspected to inhibit the activity of the SMS 27 protein.Potential inhibiting compounds may for instance be monoclonal orpolyclonal antibodies against the SMS 27 protein. Such antibodies may beobtained by routine immunization protocols of suitable laboratoryanimals.

The invention may be performed in or with any microorganism carrying aSMS 27 gene or equivalent or homologue thereof. Suitable microorganismsmay be selected from bacteria, either as wild type strains, mutantstrains derived by classic mutagenesis and selection methods or asrecombinant strains. Examples of such bacteria may be, e.g.,Gluconobacter, Gluconacetobacter, Acetobacter, Ketogulonicigenium,Pantoea, Rhizobium, Sinohrizobium, such as Sinorhizobium meliloti,Bradyrhizobium, such as Bradyrhizobium japonicum, Roseobacter,Ralstonia, Pseudomonas, such as, e.g., Pseudomonas putida, andEscherichia, such as, e.g., Escherichia coli. Preferred areGluconobacter or Acetobacter aceti, such as for instance G. oxydans, G.cerinus, G. frateurii, A. aceti subsp. xylinum or A. aceti subsp.orleanus, preferably G. oxydans IFO 3293, and their derivatives carryinggenes involved in Vitamin C production pathways and the adjacent regionswhere the SMS 27 gene or its equivalent might be located.

Microorganisms which can be used for the present invention may bepublicly available from different sources, e.g., Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1B, D-38124Braunschweig, Germany, American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108 USA or Culture Collection Division, NITEBiological Resource Center, 2-5-8, Kazusakamatari, Kisarazushi, Chiba,292-0818, Japan (formerly: Institute for Fermentation, Osaka (IFO),17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan).Examples of preferred bacteria deposited with IFO are for instanceGluconobacter oxydans (formerly known as G. melanogenus) IFO 3293,Gluconobacter oxydans (formerly known as G. melanogenus) IFO 3292,Gluconobacter oxydans (formerly known as G. rubiginosus) IFO 3244,Gluconobacter frateurii (formerly known as G. industrius) IFO 3260,Gluconobacter cerinus IFO 3266, Gluconobacter oxydans IFO 3287, andAcetobacter aceti subsp. orleanus IFO 3259, which were all deposited onApr. 5, 1954; Acetobacter aceti subsp. xylinum IFO 13693 deposited onOct. 22, 1975, and Acetobacter aceti subsp. xylinum IFO 13773 depositedon Dec. 8, 1977. Strain Acetobacter sp. ATCC 15164, which is also anexample of a preferred bacterium, was deposited with ATCC.

A microorganism as of the present invention may carry furthermodifications either on the DNA or protein level (see above), as long assuch modification has a direct impact on the yield, production and/orefficiency of the direct production of Vitamin C and/or 2-KGA fromsubstrates like e.g. D-sorbitol or L-sorbose. Such further modificationsmay for instance affect other genes encoding SMS proteins as describedabove, in particular genes encoding membrane-bound L-sorbosonedehydrogenases, such as L-sorbosone dehydrogenase SNDHai, membrane-boundPQQ bound D-sorbitol dehydrogenases and/or other genes encoding proteinsinvolved transport of sugar and/or sugar alcohols, such as e.g.exporters, in particular sorbosone exporters, preferably a gene as shownin SEQ ID NO:15. Methods of performing such modifications are known inthe art, with some examples further described herein. For the use ofSNDHai for direct production of Vitamin C as well as the nucleotide andamino acid sequence thereof we refer to WO 2005/017159 which isincorporated herein by reference.

In accordance with a further object of the present invention there isprovided the use of a polynucleotide as defined above or a microorganismwhich is genetically engineered using such polynucleotides in theproduction of Vitamin C and/or 2-KGA.

The invention also relates to processes for the expression of endogenousgenes in a microorganism, to processes for the production ofpolypeptides as defined above in a microorganism and to processes forthe production of microorganisms capable of producing Vitamin C and/or2-KGA. All these processes may comprise the step of altering amicroorganism, wherein “altering” as used herein encompasses the processfor “genetically altering” or “altering the composition of the cellculture media and/or methods used for culturing” in such a way that theyield and/or productivity of the fermentation product can be improvedcompared to the wild-type organism. As used herein, “improved yield ofVitamin C” means an increase of at least 5%, 10%, 25%, 30%, 40%, 50%,75%, 100%, 200% or even more than 500%, compared to a wild-typemicroorganism, i.e. a microorganism which is not genetically altered.

The term “genetically engineered” or “genetically altered” means thescientific alteration of the structure of genetic material in a livingorganism. It involves the production and use of recombinant DNA. More inparticular it is used to delineate the genetically engineered ormodified organism from the naturally occurring organism. Geneticengineering may be done by a number of techniques known in the art, suchas e.g. gene replacement, gene amplification, gene disruption,transfection, transformation using plasmids, viruses, or other vectors.A genetically modified organism, e.g. genetically modifiedmicroorganism, is also often referred to as a recombinant organism, e.g.recombinant microorganism.

In accordance with still another aspect of the invention there isprovided a process for the production of Vitamin C and/or 2-KGA bydirect fermentation.

Several substrates may be used as a carbon source in a process of thepresent invention, i.e. a process for direct conversion of a givensubstrate into Vitamin C such as e.g. mentioned above. Particularlysuited carbon sources are those that are easily obtainable from theD-glucose or D-sorbitol metabolization pathway such as, for example,D-glucose, D-sorbitol, L-sorbose, L-sorbosone, 2-keto-L-gluconate,D-gluconate, 2-keto-D-gluconate or 2,5-diketo-gluconate. Preferably, thesubstrate is selected from for instance D-glucose, D-sorbitol, L-sorboseor L-sorbosone, more preferably from D-glucose, D-sorbitol or L-sorbose,and most preferably from D-sorbitol, L-sorbose or L-sorbosone. The term“substrate” and “production substrate” in connection with the aboveprocess using a microorganism is used interchangeably herein.

A medium as used herein for the above process using a microorganism maybe any suitable medium for the production of Vitamin C and/or 2-KGA.Typically, the medium is an aqueous medium comprising for instancesalts, substrate(s), and a certain pH. The medium in which the substrateis converted into Vitamin C and/or 2-KGA is also referred to as theproduction medium.

“Fermentation” or “production” or “fermentation process” as used hereinmay be the use of growing cells using media, conditions and proceduresknown to the skilled person, or the use of non-growing so-called restingcells, after they have been cultivated by using media, conditions andprocedures known to the skilled person, under appropriate conditions forthe conversion of suitable substrates into desired products such asVitamin C and/or 2-KGA. Preferably, resting cells are used for theproduction of Vitamin C. An example of such process for the productionof Vitamin C is described in WO 2005/017159 (as incorporated herein byreference). Preferably, 2-KGA is produced using growing cells (see, e.g.EP 0 518 136 B1).

The term “direct fermentation”, “direct production”, “direct conversion”and the like is intended to mean that a microorganism is capable of theconversion of a certain substrate into the specified product by means ofone or more biological conversion steps, without the need of anyadditional chemical conversion step. For instance, the term “directconversion of D-sorbitol into Vitamin C” is intended to describe aprocess wherein a microorganism is producing Vitamin C and whereinD-sorbitol is offered as a carbon source without the need of anintermediate chemical conversion step. A single microorganism capable ofdirectly fermenting Vitamin C is preferred. Said microorganism iscultured under conditions which allow such conversion from the substrateas defined above.

In connection with the above process using a microorganism it isunderstood that the above-mentioned microorganisms also include synonymsor basonyms of such species having the same physiological properties, asdefined by the International Code of Nomenclature of Prokaryotes. Thenomenclature of the microorganisms as used herein is the one officiallyaccepted (at the filing date of the priority application) by theInternational Committee on Systematics of Prokaryotes and theBacteriology and Applied Microbiology Division of the InternationalUnion of Microbiological Societies, and published by its officialpublication vehicle International Journal of Systematic and EvolutionaryMicrobiology (IJSEM). A particular reference is made to Urbance et al.,IJSEM (2001) vol 51:1059-1070, with a corrective notification on IJSEM(2001) vol 51:1231-1233, describing the taxonomically reclassificationof G. oxydans DSM 4025 as Ketogulonicigenium vulgare.

As used herein, resting cells refer to cells of a microorganism whichare for instance viable but not actively growing, or which are growingat low specific growth rates, for instance, growth rates that are lowerthan 0.02 h⁻¹, preferably lower than 0.01 h⁻¹. Cells which show theabove growth rates are said to be in a “resting cell mode”.

The process of the present invention as above using a microorganism maybe performed in different steps or phases: preferably, the microorganismis cultured in a first step (also referred to as step (a) or growthphase) under conditions which enable growth. This phase is terminated bychanging of the conditions such that the growth rate of themicroorganism is reduced leading to resting cells, also referred to asstep (b), followed by the production of Vitamin C from the substrateusing the (b), also referred to as production phase.

Growth and production phase as performed in the above process using amicroorganism may be performed in the same vessel, i.e., only onevessel, or in two or more different vessels, with an optional cellseparation step between the two phases. The produced Vitamin C can berecovered from the cells by any suitable means. Recovering means forinstance that the produced Vitamin C may be separated from theproduction medium. Optionally, the thus produced Vitamin C may befurther processed.

For the purpose of the present invention relating to the above processusing a microorganism, the terms “growth phase”, “growing step”, “growthstep” and “growth period” are used interchangeably herein. The sameapplies for the terms “production phase”, “production step”, “productionperiod”.

One way of performing the above process using a microorganism as of thepresent invention may be a process wherein the microorganism is grown ina first vessel, the so-called growth vessel, as a source for the restingcells, and at least part of the cells are transferred to a secondvessel, the so-called production vessel. The conditions in theproduction vessel may be such that the cells transferred from the growthvessel become resting cells as defined above. Vitamin C is produced inthe second vessel and recovered therefrom.

In connection with the above process using a microorganism, in oneaspect, the growing step can be performed in an aqueous medium, i.e. thegrowth medium, supplemented with appropriate nutrients for growth underaerobic conditions. The cultivation may be conducted, for instance, inbatch, fed-batch, semi-continuous or continuous mode. The cultivationperiod may vary depending on for instance the host, pH, temperature andnutrient medium to be used, and may be for instance about 10 h to about10 days, preferably about 1 to about 10 days, more preferably about 1 toabout 5 days when run in batch or fed-batch mode, depending on themicroorganism. If the cells are grown in continuous mode, the residencetime may be for instance from about 2 to about 100 h, preferably fromabout 2 to about 50 h, depending on the microorganism. If themicroorganism is selected from bacteria, the cultivation may beconducted for instance at a pH of about 3.0 to about 9.0, preferablyabout 4.0 to about 9.0, more preferably about 4.0 to about 8.0, evenmore preferably about 5.0 to about 8.0. If algae or yeast are used, thecultivation may be conducted, for instance, at a pH below about 7.0,preferably below about 6.0, more preferably below about 5.5, and mostpreferably below about 5.0. A suitable temperature range for carryingout the cultivation using bacteria may be for instance from about 13° C.to about 40° C., preferably from about 18° C. to about 37° C., morepreferably from about 13° C. to about 36° C., and most preferably fromabout 18° C. to about 33° C. If algae or yeast are used, a suitabletemperature range for carrying out the cultivation may be for instancefrom about 15° C. to about 40° C., preferably from about 20° C. to about45° C., more preferably from about 25° C. to about 40° C., even morepreferably from about 25° C. to about 38° C., and most preferably fromabout 30° C. to about 38° C. The culture medium for growth usually maycontain such nutrients as assimilable carbon sources, e.g., glycerol,D-mannitol, D-sorbitol, L-sorbose, erythritol, ribitol, xylitol,arabitol, inositol, dulcitol, D-ribose, D-fructose, D-glucose, sucrose,and ethanol, preferably L-sorbose, D-glucose, D-sorbitol, D-mannitol,glycerol and ethanol; and digestible nitrogen sources such as organicsubstances, e.g., peptone, yeast extract and amino acids. The media maybe with or without urea and/or corn steep liquor and/or baker's yeast.Various inorganic substances may also be used as nitrogen sources, e.g.,nitrates and ammonium salts. Furthermore, the growth medium, usually maycontain inorganic salts, e.g., magnesium sulfate, manganese sulfate,potassium phosphate, and calcium carbonate. Cells obtained using theprocedures described above can then be further incubated at essentiallythe same modes, temperature and pH conditions as described above, in thepresence of substrates such as D-sorbitol, L-sorbose, or D-glucose, insuch a way that they convert these substrates directly into Vitamin Cand/or 2-KGA. Incubation can be done in a nitrogen-rich medium,containing, for example, organic nitrogen sources, e.g., peptone, yeastextract, baker's yeast, urea, amino acids, and corn steep liquor, orinorganic nitrogen sources, e.g., nitrates and ammonium salts, in whichcase cells will be able to further grow while producing Vitamin C and/or2-KGA. Alternatively, incubation can be done in a nitrogen-poor medium,in which case cells will not grow substantially, and will be in aresting cell mode, or biotransformation mode. In all cases, theincubation medium may also contain inorganic salts, e.g., magnesiumsulfate, manganese sulfate, potassium phosphate, and calcium chloride.

In connection with the above process using a microorganism, in thegrowth phase the specific growth rates are for instance at least 0.02h⁻¹. For cells growing in batch, fed-batch or semi-continuous mode, thegrowth rate depends on for instance the composition of the growthmedium, pH, temperature, and the like. In general, the growth rates maybe for instance in a range from about 0.05 to about 0.2 h⁻¹, preferablyfrom about 0.06 to about 0.15 h⁻¹, and most preferably from about 0.07to about 0.13 h⁻¹.

In another aspect of the above process using a microorganism, restingcells may be provided by cultivation of the respective microorganism onagar plates thus serving as growth vessel, using essentially the sameconditions, e.g., cultivation period, pH, temperature, nutrient mediumas described above, with the addition of agar agar.

In connection with the above process using a microorganism, if thegrowth and production phase are performed in two separate vessels, thenthe cells from the growth phase may be harvested or concentrated andtransferred to a second vessel, the so-called production vessel. Thisvessel may contain an aqueous medium supplemented with any applicableproduction substrate that can be converted to Vitamin C by the cells.Cells from the growth vessel can be harvested or concentrated by anysuitable operation, such as for instance centrifugation, membranecrossflow ultrafiltration or microfiltration, filtration, decantation,flocculation. The cells thus obtained may also be transferred to theproduction vessel in the form of the original broth from the growthvessel, without being harvested, concentrated or washed, i.e. in theform of a cell suspension. In a preferred embodiment, the cells aretransferred from the growth vessel to the production vessel in the formof a cell suspension without any washing or isolating step in-between.

Thus, in a preferred embodiment of the above process using amicroorganism step (a) and (c) of the process of the present inventionas described above are not separated by any washing and/or separationstep.

In connection with the above process using a microorganism, if thegrowth and production phase are performed in the same vessel, cells maybe grown under appropriate conditions to the desired cell densityfollowed by a replacement of the growth medium with the productionmedium containing the production substrate. Such replacement may be, forinstance, the feeding of production medium to the vessel at the sametime and rate as the withdrawal or harvesting of supernatant from thevessel. To keep the resting cells in the vessel, operations for cellrecycling or retention may be used, such as for instance cell recyclingsteps. Such recycling steps, for instance, include but are not limitedto methods using centrifuges, filters, membrane crossflowmicrofiltration of ultrafiltration steps, membrane reactors,flocculation, or cell immobilization in appropriate porous, non-porousor polymeric matrixes. After a transition phase, the vessel is broughtto process conditions under which the cells are in a resting cell modeas defined above, and the production substrate is efficiently convertedinto Vitamin C.

The aqueous medium in the production vessel as used for the productionstep in connection with the above process using a microorganism,hereinafter called production medium, may contain only the productionsubstrate(s) to be converted into Vitamin C, or may contain for instanceadditional inorganic salts, e.g., sodium chloride, calcium chloride,magnesium sulfate, manganese sulfate, potassium phosphate, calciumphosphate, and calcium carbonate. The production medium may also containdigestible nitrogen sources such as for instance organic substances,e.g., peptone, yeast extract, urea, amino acids, and corn steep liquor,and inorganic substances, e.g. ammonia, ammonium sulfate, and sodiumnitrate, at such concentrations that the cells are kept in a restingcell mode as defined above. The medium may be with or without ureaand/or corn steep liquor and/or baker's yeast. The production step maybe conducted for instance in batch, fed-batch, semi-continuous orcontinuous mode. In case of fed-batch, semi-continuous or continuousmode, both cells from the growth vessel and production medium can be fedcontinuously or intermittently to the production vessel at appropriatefeed rates. Alternatively, only production medium may be fedcontinuously or intermittently to the production vessel, while the cellscoming from the growth vessel are transferred at once to the productionvessel. The cells coming from the growth vessel may be used as a cellsuspension within the production vessel or may be used as for instanceflocculated or immobilized cells in any solid phase such as porous orpolymeric matrixes. The production period, defined as the period elapsedbetween the entrance of the substrate into the production vessel and theharvest of the supernatant containing Vitamin C, the so-called harveststream, can vary depending for instance on the kind and concentration ofcells, pH, temperature and nutrient medium to be used, and is preferablyabout 2 to about 100 h. The pH and temperature can be different from thepH and temperature of the growth step, but is essentially the same asfor the growth step.

In a preferred embodiment of the above process using a microorganism,the production step is conducted in continuous mode, meaning that afirst feed stream containing the cells from the growth vessel and asecond feed stream containing the substrate is fed continuously orintermittently to the production vessel. The first stream may eithercontain only the cells isolated/separated from the growth medium or acell suspension, coming directly from the growth step, i.e. cellssuspended in growth medium, without any intermediate step of cellseparation, washing and/or isolating. The second feed stream as hereindefined may include all other feed streams necessary for the operationof the production step, e.g. the production medium comprising thesubstrate in the form of one or several different streams, water fordilution, and base for pH control.

In connection with the above process using a microorganism, when bothstreams are fed continuously, the ratio of the feed rate of the firststream to feed rate of the second stream may vary between about 0.01 andabout 10, preferably between about 0.01 and about 5, most preferablybetween about 0.02 and about 2. This ratio is dependent on theconcentration of cells and substrate in the first and second stream,respectively.

Another way of performing the process as above using a microorganism ofthe present invention may be a process using a certain cell density ofresting cells in the production vessel. The cell density is measured asabsorbance units (optical density) at 600 nm by methods known to theskilled person. In a preferred embodiment, the cell density in theproduction step is at least about 10, more preferably between about 10and about 200, even more preferably between about 15 and about 200, evenmore preferably between about 15 to about 120, and most preferablybetween about 20 and about 120.

In connection with the above process using a microorganism, in order tokeep the cells in the production vessel at the desired cell densityduring the production phase as performed, for instance, in continuous orsemi-continuous mode, any means known in the art may be used, such asfor instance cell recycling by centrifugation, filtration, membranecrossflow ultrafiltration of microfiltration, decantation, flocculation,cell retention in the vessel by membrane devices or cell immobilization.Further, in case the production step is performed in continuous orsemi-continuous mode and cells are continuously or intermittently fedfrom the growth vessel, the cell density in the production vessel may bekept at a constant level by, for instance, harvesting an amount of cellsfrom the production vessel corresponding to the amount of cells beingfed from the growth vessel.

In connection with the above process using a microorganism, the producedVitamin C contained in the so-called harvest stream isrecovered/harvested from the production vessel. The harvest stream mayinclude, for instance, cell-free or cell-containing aqueous solutioncoming from the production vessel, which contains Vitamin C as a resultof the conversion of production substrate by the resting cells in theproduction vessel. Cells still present in the harvest stream may beseparated from the Vitamin C by any operations known in the art, such asfor instance filtration, centrifugation, decantation, membrane crossflowultrafiltration or microfiltration, tangential flow ultrafiltration ormicrofiltration or dead end filtration. After this cell separationoperation, the harvest stream is essentially free of cells.

In a further aspect, the process of the present invention may becombined with further steps of separation and/or purification of theproduced Vitamin C from other components contained in the harveststream, i.e., so-called downstream processing steps. These steps mayinclude any means known to a skilled person, such as, for instance,concentration, crystallization, precipitation, adsorption, ion exchange,electrodialysis, bipolar membrane electrodialysis and/or reverseosmosis. Vitamin C may be further purified as the free acid form or anyof its known salt forms by means of operations such as for instancetreatment with activated carbon, ion exchange, adsorption and elution,concentration, crystallization, filtration and drying. Specifically, afirst separation of Vitamin C from other components in the harveststream might be performed by any suitable combination or repetition of,for instance, the following methods: two- or three-compartmentelectrodialysis, bipolar membrane electrodialysis, reverse osmosis oradsorption on, for instance, ion exchange resins or non-ionic resins. Ifthe resulting form of Vitamin C is a salt of L-ascorbic acid, conversionof the salt form into the free acid form may be performed by forinstance bipolar membrane electrodialysis, ion exchange, simulatedmoving bed chromatographic techniques, and the like. Combination of thementioned steps, e.g., electrodialysis and bipolar membraneelectrodialysis into one step might be also used as well as combinationof the mentioned steps e.g. several steps of ion exchange by usingsimulated moving bed chromatographic methods. Any of these proceduresalone or in combination constitute a convenient means for isolating andpurifying the product, i.e. Vitamin C. The product thus obtained mayfurther be isolated in a manner such as, e.g. by concentration,crystallization, precipitation, washing and drying of the crystalsand/or further purified by, for instance, treatment with activatedcarbon, ion exchange and/or re-crystallization.

In a preferred embodiment, Vitamin C is purified from the harvest streamby a series of downstream processing steps as described above withouthaving to be transferred to a non-aqueous solution at any time of thisprocessing, i.e. all steps are performed in an aqueous environment. Suchpreferred downstream processing procedure may include for instance theconcentration of the harvest stream coming from the production vessel bymeans of two- or three-compartment electrodialysis, conversion ofVitamin C in its salt form present in the concentrated solution into itsacid form by means of bipolar membrane electrodialysis and/or ionexchange, purification by methods such as for instance treatment withactivated carbon, ion exchange or non-ionic resins, followed by afurther concentration step and crystallization. These crystals can beseparated, washed and dried. If necessary, the crystals may be againre-solubilized in water, treated with activated carbon and/or ionexchange resins and recrystallized. These crystals can then beseparated, washed and dried.

Advantageous embodiments of the invention become evident from thedependent claims. These and other aspects and embodiments of the presentinvention should be apparent to those skilled in the art from theteachings herein.

The sequence of the gene comprising a nucleotide sequence according toSEQ ID NO:1 encoding a SMS 27 protein was determined by sequencing agenomic clone obtained from Gluconobacter oxydans IFO 3293.

The invention also relates to a polynucleotide encoding at least abiologically active fragment or derivative of a SMS 27 polypeptide asshown in SEQ ID NO:2.

As used herein, “biologically active fragment or derivative” means apolypeptide which retains essentially the same biological function oractivity as the polypeptide shown in SEQ ID NO:2. Examples of biologicalactivity may for instance be enzymatic activity, signaling activity orantibody reactivity. The term “same biological function” or “functionalequivalent” as used herein means that the protein has essentially thesame biological activity, e.g. enzymatic, signaling antibody reactivitytranscriptionally regulator, as a polypeptide shown in SEQ ID NO:2.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living microorganism is not isolated, but thesame polynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.

An isolated polynucleotide or nucleic acid as used herein may be a DNAor RNA that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (one on the 5′-end andone on the 3′-end) in the naturally occurring genome of the organismfrom which it is derived. Thus, in one embodiment, a nucleic acidincludes some or all of the 5′-non-coding (e.g., promoter) sequencesthat are immediately contiguous to the coding sequence. The term“isolated polynucleotide” therefore includes, for example, a recombinantDNA that is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences. It also includes a recombinant DNA thatis part of a hybrid gene encoding an additional polypeptide that issubstantially free of cellular material, viral material, or culturemedium (when produced by recombinant DNA techniques), or chemicalprecursors or other chemicals (when chemically synthesized). Moreover,an “isolated nucleic acid fragment” is a nucleic acid fragment that isnot naturally occurring as a fragment and would not be found in thenatural state.

As used herein, the terms “polynucleotide”, “gene” and “recombinantgene” refer to nucleic acid molecules which may be isolated fromchromosomal DNA, which include an open reading frame encoding a protein,e.g. G. oxydans IFO 3293 SMS proteins. A polynucleotide may include apolynucleotide sequence as shown in SEQ ID NO:1 or fragments thereof andregions upstream and downstream of the gene sequences which may include,for example, promoter regions, regulator regions and terminator regionsimportant for the appropriate expression and stabilization of thepolypeptide derived thereof.

A gene may include coding sequences, non-coding sequences such as forinstance untranslated sequences located at the 3′- and 5′-ends of thecoding region of a gene, and regulatory sequences. Moreover, a generefers to an isolated nucleic acid molecule as defined herein. It isfurthermore appreciated by the skilled person that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of SMSproteins may exist within a population, e.g., the Gluconobacter oxydanspopulation. Such genetic polymorphism in the SMS 27 gene may exist amongindividuals within a population due to natural variation or in cellsfrom different populations. Such natural variations can typically resultin 1-5% variance in the nucleotide sequence of the SMS 27 gene. Any andall such nucleotide variations and the resulting amino acid polymorphismin SMS 27 are the result of natural variation and that do not alter thefunctional activity of SMS proteins are intended to be within the scopeof the invention.

As used herein, the terms “polynucleotide” or “nucleic acid molecule”are intended to include DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleic acidmay be synthesized using oligonucleotide analogs or derivatives (e.g.,inosine or phosphorothioate nucleotides). Such oligonucleotides may beused, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases.

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Thespecific sequences disclosed herein may be readily used to isolate thecomplete gene from a recombinant or non-recombinant microorganismcapable of converting a given carbon source directly into Vitamin Cand/or 2-KGA, in particular Gluconobacter oxydans, preferablyGluconobacter oxydans IFO 3293 which in turn may easily be subjected tofurther sequence analyses thereby identifying sequencing errors.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer and all amino acid sequences of polypeptides encoded by DNAmolecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence may be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

The person skilled in the art is capable of identifying such erroneouslyidentified bases and knows how to correct for such errors.

A nucleic acid molecule according to the invention may comprise only aportion or a fragment of the nucleic acid sequence provided by thepresent invention, such as for instance the sequence shown in SEQ IDNO:1, for example a fragment which may be used as a probe or primer suchas for instance SEQ ID NO:3 or SEQ ID NO:4 or a fragment encoding aportion of a protein according to the invention. The nucleotide sequencedetermined from the cloning of the SMS 27 gene allows for the generationof probes and primers designed for use in identifying and/or cloningother SMS 27 family members, as well as SMS 27 homologues from otherspecies. The probe/primer typically comprises substantially purifiedoligonucleotides which typically comprises a region of nucleotidesequence that hybridizes preferably under highly stringent conditions toat least about 12 or 15, preferably about 18 or 20, more preferablyabout 22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60,65, or 75 or more consecutive nucleotides of a nucleotide sequence shownin SEQ ID NO:1 or a fragment or derivative thereof.

A nucleic acid molecule encompassing all or a portion of the nucleicacid sequence of SEQ ID NO:1 may be also isolated by the polymerasechain reaction (PCR) using synthetic oligonucleotide primers designedbased upon the sequence information contained herein.

A nucleic acid of the invention may be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid thus amplified may be cloned into anappropriate vector and characterized by DNA sequence analysis.

Fragments of a polynucleotide according to the invention may alsocomprise polynucleotides not encoding functional polypeptides. Suchpolynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether theyencode functional or non-functional polypeptides, may be used ashybridization probes or polymerase chain reaction (PCR) primers. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having a SMS 27 activity include, inter alia, (1) isolatingthe gene encoding the protein of the present invention, or allelicvariants thereof from a cDNA library, e.g., from other organisms thanGluconobacter oxydans and (2) Northern blot analysis for detectingexpression of mRNA of said protein in specific cells or (3) use inenhancing and/or improving the function or activity of homologous SMS 27genes in said other organisms.

Probes based on the nucleotide sequences provided herein may be used todetect transcripts or genomic sequences encoding the same or homologousproteins for instance in other organisms. Nucleic acid moleculescorresponding to natural variants and non-G. oxydans homologues of theG. oxydans SMS 27 DNA of the invention which are also embraced by thepresent invention may be isolated based on their homology to the G.oxydans SMS 27 nucleic acid disclosed herein using the G. oxydans DNA,or a portion thereof, as a hybridization probe according to standardhybridization techniques, preferably under highly stringenthybridization conditions.

In preferred embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme cofactor.

Homologous gene sequences may be isolated, for example, by performingPCR using two degenerate oligonucleotide primer pools designed on thebasis of nucleotide sequences as taught herein.

The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from strains known or suspected toexpress a polynucleotide according to the invention. The PCR product maybe subcloned and sequenced to ensure that the amplified sequencesrepresent the sequences of a new nucleic acid sequence as describedherein, or a functional equivalent thereof.

The PCR fragment may then be used to isolate a full length cDNA clone bya variety of known methods. For example, the amplified fragment may belabeled and used to screen a bacteriophage or cosmid cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary.

PCR technology can also be used to isolate full-length cDNA sequencesfrom other organisms. For example, RNA may be isolated, followingstandard procedures, from an appropriate cellular or tissue source. Areverse transcription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′-end of the amplifiedfragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid may then be “tailed” (e.g., with guanines)using a standard terminal transferase reaction, the hybrid may bedigested with RNaseH, and second strand synthesis may then be primed(e.g., with a poly-C primer). Thus, cDNA sequences upstream of theamplified fragment may easily be isolated. For a review of usefulcloning strategies, see e.g., Sambrook et al., supra; and Ausubel etal., supra.

Also, nucleic acids encoding other SMS 27 family members, which thushave a nucleotide sequence that differs from a nucleotide sequenceaccording to SEQ ID NO:1, are within the scope of the invention.Moreover, nucleic acids encoding SMS 27 proteins from different specieswhich thus may have a nucleotide sequence which differs from anucleotide sequence shown in SEQ ID NO:1 are within the scope of theinvention.

The invention also relates to an isolated polynucleotide hybridisableunder stringent conditions, preferably under highly stringentconditions, to a polynucleotide as of the present invention, such as forinstance a polynucleotide shown in SEQ ID NO:1. Advantageously, suchpolynucleotide may be obtained from a microorganism capable ofconverting a given carbon source directly into Vitamin C, in particularGluconobacter oxydans, preferably Gluconobacter oxydans IFO 3293.

As used herein, the term “hybridizing” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least about 50%, at least about 60%, at least about 70%,more preferably at least about 80%, even more preferably at least about85% to 90%, most preferably at least 95% homologous to each othertypically remain hybridized to each other.

In one embodiment, a nucleic acid of the invention is at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shownin SEQ ID NO:1 or the complement thereof.

A preferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 1×SSC, 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 60° C. and even more preferablyat 65° C.

Highly stringent conditions include incubations at 42° C. for a periodof several days, such as 2-4 days, using a labeled DNA probe, such as adigoxigenin (DIG)-labeled DNA probe, followed by one or more washes in2×SSC, 0.1% SDS at room temperature and one or more washes in 0.5×SSC,0.1% SDS or 0.1×SSC, 0.1% SDS at 65-68° C. In particular, highlystringent conditions include, for example, 2 h to 4 days incubation at42° C. using a DIG-labeled DNA probe (prepared by e.g. using a DIGlabeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in asolution such as DigEasyHyb solution (Roche Diagnostics GmbH) with orwithout 100 μg/ml salmon sperm DNA, or a solution comprising 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodiumdodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (RocheDiagnostics GmbH), followed by washing the filters twice for 5 to 15minutes in 2×SSC and 0.1% SDS at room temperature and then washing twicefor 15-30 minutes in 0.5×SSC and 0.1% SDS or 0.1×SSC and 0.1% SDS at65-68° C.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under preferably highly stringent conditions to a nucleotidesequence of the invention corresponds to a naturally-occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein). In oneembodiment, the nucleic acid encodes a natural G. oxydans SMS 27protein.

The skilled artisan will know which conditions to apply for stringentand highly stringent hybridization conditions. Additional guidanceregarding such conditions is readily available in the art, for example,in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, CurrentProtocols in Molecular Biology, (John Wiley & Sons, N.Y.). Of course, apolynucleotide which hybridizes only to a poly (A) sequence (such as the3′-terminal poly (A) tract of mRNAs), or to a complementary stretch of T(or U) residues, would not be included in a polynucleotide of theinvention used to specifically hybridize to a portion of a nucleic acidof the invention, since such a polynucleotide would hybridize to anynucleic acid molecule containing a poly (A) stretch or the complementthereof (e.g., practically any double-stranded cDNA clone).

In a typical approach, genomic DNA or cDNA libraries constructed fromother organisms, e.g. microorganisms capable of converting a givencarbon source directly into Vitamin C, in particular other Gluconobacterspecies may be screened.

For example, Gluconobacter strains may be screened for homologouspolynucleotides by Southern and/or Northern blot analysis. Upondetection of transcripts homologous to polynucleotides according to theinvention, DNA libraries may be constructed from RNA isolated from theappropriate strain, utilizing standard techniques well known to those ofskill in the art. Alternatively, a total genomic DNA library may bescreened using a probe hybridisable to a polynucleotide according to theinvention.

A nucleic acid molecule of the present invention, such as for instance anucleic acid molecule shown in SEQ ID NO:1 or a fragment or derivativethereof, may be isolated using standard molecular biology techniques andthe sequence information provided herein. For example, using all orportion of the nucleic acid sequence shown in SEQ ID NO:1 as ahybridization probe, nucleic acid molecules according to the inventionmay be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

Furthermore, oligonucleotides corresponding to or hybridisable tonucleotide sequences according to the invention may be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

The terms “homology” or “percent identity” are used interchangeablyherein. For the purpose of this invention, it is defined here that inorder to determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps may be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical positions/totalnumber of positions (i.e., overlapping positions)×100). Preferably, thetwo sequences are the same length.

The skilled person will be aware of the fact that several differentcomputer programs are available to determine the homology between twosequences. For instance, a comparison of sequences and determination ofpercent identity between two sequences may be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.accelrys.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and alength weight of 1, 2, 3, 4, 5 or 6. The skilled person will appreciatethat all these different parameters will yield slightly differentresults but that the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

In yet another embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available at http://www.accelrys.com), using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of1, 2, 3, 4, 5 or 6. In another embodiment, the percent identity betweentwo amino acid or nucleotide sequences is determined using the algorithmof E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989) which has beenincorporated into the ALIGN program (version 2.0) (available athttp://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention mayfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches may be performed using the BLASTN and BLASTXprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches may be performed with the BLASTNprogram, score=100, word length=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the present invention. BLASTprotein searches may be performed with the BLASTX program, score=50,word length=3 to obtain amino acid sequences homologous to the proteinmolecules of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST may be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) may be used. Seehttp://www.ncbi.nlm.nih.gov.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is the complementof a nucleotide sequence as of the present invention, such as forinstance the sequence shown in SEQ ID NO:1. A nucleic acid molecule,which is complementary to a nucleotide sequence disclosed herein, is onethat is sufficiently complementary to a nucleotide sequence shown in SEQID NO:1 such that it may hybridize to said nucleotide sequence therebyforming a stable duplex.

In a further preferred embodiment, a nucleic acid of the invention asshown in SEQ ID NO:1 or the complement thereof contains at least onemutation leading to a gene product with modified function/activity. Theat least one mutation may be introduced by methods described herein. Inone aspect, the at least one mutation leads to a SMS 27 protein whosefunction compared to the wild type counterpart is completely orpartially destroyed. Methods for introducing such mutations are wellknown in the art.

The term “reduction” of activity as used herein encompasses decreasingactivity of one or more polypeptides in the producing organism, which inturn are encoded by the corresponding polynucleotides described herein.There are a number of methods available in the art to accomplishreduction of activity of a given protein, in this case the SMS 27protein. In general, the specific activity of a protein may be decreasedor the copy number of the protein may be decreased.

To facilitate such a decrease, the copy number of the genescorresponding to the polynucleotides described herein may be decreased,such as for instance by underexpression or disruption of a gene. A geneis said to be “underexpressed” if the level of transcription of saidgene is reduced in comparison to the wild type gene. This may bemeasured by for instance Northern blot analysis quantifying the amountof mRNA as an indication for gene expression. As used herein, a gene isunderexpressed if the amount of generated mRNA is decreased by at least1%, 2%, 5% 10%, 25%, 50%, 75%, 100%, 200% or even more than 500%,compared to the amount of mRNA generated from a wild-type gene.Alternatively, a weak promoter may be used to direct the expression ofthe polynucleotide. In another embodiment, the promoter, regulatoryregion and/or the ribosome binding site upstream of the gene can bealtered to achieve the down-expression. The expression may also bereduced by decreasing the relative half-life of the messenger RNA. Inanother embodiment, the activity of the polypeptide itself may bedecreased by employing one or more mutations in the polypeptide aminoacid sequence, which decrease the activity. For example, altering theaffinity of the polypeptide for its corresponding substrate may resultin reduced activity. Likewise, the relative half-life of the polypeptidemay be decreased. In either scenario, that being reduced gene expressionor reduced activity, the reduction may be achieved by altering thecomposition of the cell culture media and/or methods used for culturing.“Reduced expression” or “reduced activity” as used herein means adecrease of at least 5%, 10%, 25%, 50%, 75%, 100%, 200% or even morethan 500%, compared to a wild-type protein, polynucleotide, gene; or theactivity and/or the concentration of the protein present before thepolynucleotides or polypeptides are reduced. The activity of the SMS 27protein may also be reduced by contacting the protein with a specific orgeneral inhibitor of its activity. The terms “reduced activity”,“decreased or abolished activity” are used interchangeably herein.

Another aspect of the invention pertains to vectors, containing anucleic acid encoding a protein according to the invention or afunctional equivalent or portion thereof. As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated. Another type of vector isa viral vector, wherein additional DNA segments may be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication). Other vectors are integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome.

The recombinant vectors of the invention comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vector includesone or more regulatory sequences, selected on the basis of the hostcells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operatively linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., attenuator). Such regulatory sequences are described,for example, in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive or inducibleexpression of a nucleotide sequence in many types of host cells andthose which direct expression of the nucleotide sequence only in acertain host cell (e.g. tissue-specific regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention may be introduced into hostcells to thereby produce proteins or peptides, encoded by nucleic acidsas described herein, including, but not limited to, mutant proteins,fragments thereof, variants or functional equivalents thereof, andfusion proteins, encoded by a nucleic acid as described herein, e.g.,SMS 27 proteins, mutant forms of SMS 27 proteins, fusion proteins andthe like.

The recombinant expression vectors of the invention may be designed forexpression of SMS 27 proteins in a suitable microorganism. For example,a protein according to the invention may be expressed in bacterial cellssuch as strains belonging to the genera Gluconobacter, Gluconacetobacteror Acetobacter. Expression vectors useful in the present inventioninclude chromosomal-, episomal- and virus-derived vectors e.g., vectorsderived from bacterial plasmids, bacteriophage, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids.

The DNA insert may be operatively linked to an appropriate promoter,which may be either a constitutive or inducible promoter. The skilledperson will know how to select suitable promoters. The expressionconstructs may contain sites for transcription initiation, termination,and, in the transcribed region, a ribosome binding site for translation.The coding portion of the mature transcripts expressed by the constructsmay preferably include an initiation codon at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

Vector DNA may be introduced into suitable host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation”, “transconjugation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, transduction, infection, lipofection, cationiclipidmediated transfection or electroporation. Suitable methods fortransforming or transfecting host cells may be found in Sambrook, et al.(supra), Davis et al., Basic Methods in Molecular Biology (1986) andother laboratory manuals.

In order to identify and select cells which have integrated the foreignDNA into their genome, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Preferred selectable markers includethose that confer resistance to drugs, such as kanamycin, tetracycline,ampicillin and streptomycin. A nucleic acid encoding a selectable markeris preferably introduced into a host cell on the same vector as thatencoding a protein according to the invention or can be introduced on aseparate vector such as, for example, a suicide vector, which cannotreplicate in the host cells. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

The invention provides also an isolated polypeptide having the aminoacid sequence shown in SEQ ID NO:2 or an amino acid sequence obtainableby expressing a polynucleotide of the present invention, such as forinstance a polynucleotide sequence shown in SEQ ID NO:1 in anappropriate host.

Polypeptides according to the invention may contain only conservativesubstitutions of one or more amino acids in the amino acid sequencerepresented by SEQ ID NO:2 or substitutions, insertions or deletions ofnon-essential amino acids. Accordingly, a non-essential amino acid is aresidue that may be altered in the amino acid sequences shown in SEQ IDNO:2 without substantially altering the biological function. Forexample, amino acid residues that are conserved among the proteins ofthe present invention, are predicted to be particularly unamenable toalteration. Furthermore, amino acids conserved among the proteinsaccording to the present invention and other SMS 27 proteins are notlikely to be amenable to alteration.

The term “conservative substitution” is intended to mean that asubstitution in which the amino acid residue is replaced with an aminoacid residue having a similar side chain. These families are known inthe art and include amino acids with basic side chains (e.g., lysine,arginine and histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As mentioned above, the polynucleotides of the invention may be utilizedin the genetic engineering of a suitable host cell to make it better andmore efficient in the fermentation, for example in a direct fermentationprocess for Vitamin C.

According to the invention a genetically engineered/recombinantlyproduced host cell (also referred to as recombinant cell or transformedcell) carrying such a modified polynucleotide wherein the function ofthe linked protein is significantly modified in comparison to awild-type cell such that the yield, production and/or efficiency ofproduction of one or more fermentation products such as Vitamin C isimproved. The host cell may be selected from a microorganism capable ofdirectly producing one or more fermentation products such as forinstance Vitamin C from a given carbon source, in particularGluconobacter oxydans, preferably G. oxydans IFO 3293.

A “transformed cell” or “recombinant cell” is a cell into which (or intoan ancestor of which) has been introduced, by means of recombinant DNAtechniques, a nucleic acid according to the invention, or wherein theactivity of the SMS 27 protein has been decreased or abolished. Suitablehost cells include cells of microorganisms capable of producing a givenfermentation product, e.g., converting a given carbon source directlyinto Vitamin C. In particular, these include strains from the generaPseudomonas, Pantoea, Escherichia, Corynebacterium, Ketogulonicigeniumand acetic acid bacteria like e.g., Gluconobacter, Acetobacter orGluconacetobacter, preferably Acetobacter sp., Acetobacter aceti,Gluconobacter frateurii, Gluconobacter cerinus, Gluconobacterthailandicus, Gluconobacter oxydans, more preferably G. oxydans, mostpreferably G. oxydans IFO 3293.

To improve the Vitamin C and/or 2-KGA production of a certainrecombinant host cell, SMS 27 gene expression may be inhibited in thatorganism for instance by targeting nucleotide sequences complementary tothe regulatory region of a SMS 27 nucleotide sequence (e.g., a SMS 27promoter and/or enhancers) to form triple helical structures thatprevent transcription of a SMS 27 gene in target cells. See generally,Helene, C. (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al.(1992) Ann. N.Y. Acad. Sci. 660: 27-36; and Maher, L. J. (1992)Bioassays 14 (12): 807-15.

Inhibition or prevention of gene expression may also be achieved bymodifying the SMS 27 gene, e.g., by introducing one or more mutationsinto the SMS 27gene wherein said modification leads to a SMS 27 proteinwith a function which is significantly decreased in comparison to thewild-type protein.

Therefore, in one other embodiment, the polynucleotide carrying the atleast one mutation is derived from a polynucleotide as represented bySEQ ID NO:1 or equivalents thereof

A mutation as used herein may be any mutation leading to a lessfunctional or unstable polypeptide, e.g. less functional or unstable SMS27 gene products. This may include for instance an alteration in thegenome of a microorganism, which interferes with the synthesis of SMS 27or leads to the expression of a SMS 27 protein with an altered aminoacid sequence whose function compared with the wild type counterparthaving a non-altered amino acid sequence is completely or partiallydestroyed. The interference may occur at the transcriptional,translational or post-translational level.

The alteration in the genome of the microorganism may be obtained e.g.by replacing through a single or double crossover recombination a wildtype DNA sequence by a DNA sequence containing the alteration. Forconvenient selection of transformants of the microorganism with thealteration in its genome the alteration may, e.g. be a DNA sequenceencoding an antibiotic resistance marker or a gene complementing apossible auxotrophy of the microorganism. Mutations include, but are notlimited to, deletion-insertion mutations. An example of such analteration includes a gene disruption, i.e. a perturbation of a genesuch that the product that is normally produced from this gene is notproduced in a functional form. This could be due to a complete deletion,a deletion and insertion of a selective marker, an insertion of aselective marker, a frameshift mutation, an in-frame deletion, or apoint mutation that leads to premature termination. In some of thesecases the entire mRNA for the gene is absent, in others the amount ofmRNA produced varies. In all cases the polypeptide encoded by said geneis not produced in a functional form, either absent or in a mutatedform, such as e.g. a protein having reduced activity as defined herein.

An alteration in the genome of the microorganism leading to a less ornon-functional polypeptide may also be obtained by randomly mutagenizingthe genome of the microorganism using e.g. chemical mutagens, radiationor transposons and selecting or screening for mutants which are betteror more efficient producers of one or more fermentation products.Standard methods for screening and selection are known to the skilledperson.

In a specific embodiment, it is desired to knockout the SMS 27 gene ofthe present invention, i.e., wherein its gene expression is artificiallysuppressed in order to improve the yield, productivity, and/orefficiency of production of the fermentation product when introducedinto a suitable host cell. Methods of providing knockouts as well asmicroorganisms carrying such suppressed genes are well known in the art.The suppression of the endogenous SMS 27 gene may be induced by deletingat least a part of the gene or the regulatory region thereof. As usedherein, “suppression of the gene expression” includes complete andpartial suppression, as well as suppression under specific conditionsand also suppression of the expression of either one of the two alleles.

In order to create a knockout microorganism in which the expression ofthe SMS 27 gene is artificially suppressed, first the SMS 27 gene may becloned and then a vector for homologous recombination may be constructedby using the gene to inactivate the endogenous SMS 27 gene in the targetmicroorganism. The vector for homologous recombination then contains anucleic acid sequence designed to inactivate the endogenous SMS 27 genein the target microorganism. Such a nucleic acid may be for instance anucleic acid sequence of the SMS 27 gene or the regulatory regionthereof, such as the existing flanking region of the gene to beinactivated (in cis), or existing separately (in trans), containing atleast a partial deletion, or alternatively it may be a nucleic acidsequence of the SMS 27 gene or the regulatory region thereof containingother genes. A gene which can also function as a marker is preferablyselected as the gene to be inserted into the SMS 27 gene or theregulatory region thereof. The insert genes to be used include forinstance drug-resistance genes as defined above. There is no particularlimitation on the position where the genes may be inserted in the SMS 27gene, as long as the insertion at that position results in thesuppression of the expression of the endogenous SMS 27 gene in thetarget. To avoid polar effects of the insertion, in-frame silentdeletions can be introduced by using, for example, the sacB system orlong-flanking homology PCR. These techniques are well known to theperson skilled in the art.

The aforementioned mutagenesis strategies for SMS 27 proteins may resultin increased yields of a desired compound in particular Vitamin C and/or2-KGA. This list is not meant to be limiting; variations on thesemutagenesis strategies will be readily apparent to one of ordinary skillin the art. By these mechanisms, the nucleic acid and protein moleculesof the invention may be utilized to generate microorganisms such asGluconobacter oxydans or related strains of bacteria expressing mutatedSMS 27 nucleic acid and protein molecules such that the yield,productivity, and/or efficiency of production of a desired compound suchas Vitamin C and/or 2-KGA is improved.

In connection with the above process using a microorganism, in oneaspect, the process of the present invention leads to yields of VitaminC which are at least about more than 5.7 g/l, such as 10 g/l, 20 g/l, 50g/l, 100 g/l, 200 g/l, 300 g/l, 400 g/l or more than 600 g/l. In oneembodiment, the yield of Vitamin C produced by the process of thepresent invention is in the range of from about more than 5.7 to about600 g/l. The yield of Vitamin C refers to the concentration of Vitamin Cin the harvest stream coming directly out of the production vessel, i.e.the cell-free supernatant comprising the Vitamin C.

In one aspect of the invention, microorganisms (in particular from thegenera of Gluconobacter, Gluconacetobacter and Acetobacter) are providedthat are able to directly produce Vitamin C from a suitable carbonsource like D-sorbitol and/or L-sorbose. When measured for instance in aresting cell method after an incubation period of 20 hours, theseorganisms were found to be able to produce Vitamin C directly fromD-sorbitol or L-sorbose, even up to a level of 280 mg/l and 670 mg/lrespectively. In another aspect of the invention, a microorganism isprovided capable of directly producing Vitamin C in quantities of 300mg/l when starting from D-sorbitol or more or 800 mg/l or more whenstarting from L-sorbose, respectively when for instance measured in aresting cell method after an incubation period of 20 hours. Such may beachieved by decreasing or abolishing the activity of a SMS polypeptide,preferably a SMS 27 polypeptide. The yield of Vitamin C produced fromD-sorbitol may even be as high as 400, 600, 1000 mg/l or even exceed1.5, 2, 4, 10, 20, 50 g/l. The yield of Vitamin C produced fromL-sorbose may even be as high as 1000 mg/l or even exceed 1.5, 2, 4, 10,20, 50 μl. Preferably, these amounts of Vitamin C can be achieved whenmeasured by resting cell method after an incubation period of 20 hours.

As used herein, measurement in a “resting cell method” comprises (i)growing the cells by means of any method well know to the person skilledin the art, (ii) harvesting the cells from the growth broth, and (iii)incubating the harvested cells in a medium containing the substratewhich is to be converted into the desired product, e.g. Vitamin C, underconditions where the cells do not grow any longer, i.e. there is noincrease in the amount of biomass during this so-called conversion step.A more general description of the resting cell method is described forinstance in WO 2005/017159 and in the following paragraphs.

In one aspect of the invention, microorganisms (in particular from thegenera of Gluconobacter, Gluconacetobacter and Acetobacter) are providedthat are able to directly produce 2-KGA from a suitable carbon sourcelike D-sorbitol and/or L-sorbose. When measured for instance by themethod as of Example 4, these organisms were found to be able to produce2-KGA directly from D-sorbitol or L-sorbose in amounts of about 0.5 to0.7 g/l. In another aspect of the invention, a microorganism is providedcapable of directly producing 2-KGA in quantities of about 7, 8, 9, 10g/l or more or even about 50, 60, 70, 80, 90, 100 g/l or more whenstarting from L-sorbose. Such may be achieved by decreasing or evenabolishing the activity of a SMS polypeptide, preferably a SMS 27polypeptide in G. oxydans IFO 3293.

The recombinant microorganism carrying e.g. a modified SMS 27 gene andwhich is able to produce the fermentation product in significantlyhigher yield, productivity, and/or efficiency may be cultured in anaqueous medium supplemented with appropriate nutrients under aerobicconditions as described above.

The nucleic acid molecules, polypeptides, vectors, primers, andrecombinant microorganisms described herein may be used in one or moreof the following methods: identification of Gluconobacter oxydans andrelated organisms; mapping of genomes of organisms related toGluconobacter oxydans; identification and localization of Gluconobacteroxydans sequences of interest; evolutionary studies; determination ofSMS 27 protein regions required for function; modulation of a SMS 27protein activity or function; modulation of the activity of a SMSpathway; and modulation of cellular production of a desired compound,such as Vitamin C and/or 2-KGA.

The invention provides methods for screening molecules which modulatethe activity of a SMS 27 protein, either by interacting with the proteinitself or a substrate or binding partner of the SMS 27 protein, or bymodulating the transcription or translation of a SMS 27 nucleic acidmolecule of the invention. In such methods, a microorganism expressingone or more SMS 27 proteins of the invention is contacted with one ormore test compounds, and the effect of each test compound on theactivity or level of expression of the SMS 27 protein is assessed.

In general, the biological, enzymatic or other activity of SMS proteinscan be measured by methods well known to a skilled person, such as, forexample, by incubating a cell fraction containing the SMS protein in thepresence of its substrate, electron acceptor(s) or donor(s) includingphenazine methosulfate (PMS), dichlorophenol-indophenol (DCIP), NAD,NADH, NADP, NADPH, which consumption can be directly or indirectlymeasured by photometric, colorimetric or fluorimetric methods, and otherinorganic components which might be relevant for the development of theactivity. Thus, for example, the activity of membrane-bound D-sorbitoldehydrogenase can be measured in an assay where membrane fractionscontaining this enzyme are incubated in the presence of phosphate bufferat pH 6, D-sorbitol and the artificial electron acceptors DCIP and PMS.The rate of consumption of DCIP can be measured at 600 nm, and isdirectly proportional to the D-sorbitol dehydrogenase activity presentin the membrane fraction.

The biological, enzymatic or other activity of SMS proteins, inparticular the SMS 27 protein, can be measured by methods well known toa skilled person, such as, for example, by determining the expression ofgenes known to be under the control of the SMS 27 protein by methodsknown to those skilled in the art, such as for instance Northern Blot,transcriptional fusion analysis, microarray analysis, etc. Binding ofSMS 27 protein to target promoter regions can be demonstrated by DNAfootprint experiments, gel-shift assays and the like.

It may be evident from the above description that the fermentationproduct of the methods according to the invention may not be limited toVitamin C alone. The “desired compound” or “fermentation product” asused herein may be any natural product of Gluconobacter oxydans, whichincludes the final products and intermediates of biosynthesis pathways,such as for example L-sorbose, L-sorbosone, D-gluconate,2-keto-D-gluconate, 5-keto-D-gluconate, 2,5-diketo-D-gluconate and2-keto-L-gluconate (2-KGA), in particular the biosynthetic generation ofVitamin C.

Thus, the present invention is directed to the use of a polynucleotide,polypeptide, vector, primer and recombinant microorganism as describedherein in the production of Vitamin C and/or 2-KGA, i.e., the directconversion of a carbon source into Vitamin C and/or 2-KGA. In apreferred embodiment, a modified polynucleotide, polypeptide, vector andrecombinant microorganism as described herein is used for improving theyield, productivity, and/or efficiency of the production of Vitamin Cand/or 2-KGA.

The terms “production” or “productivity” are art-recognized and includethe concentration of the fermentation product (for example, Vitamin Cand/or 2-KGA) formed within a given time and a given fermentation volume(e.g., kg product per hour per liter). The term “efficiency ofproduction” includes the time required for a particular level ofproduction to be achieved (for example, how long it takes for the cellto attain a particular rate of output of a fermentation product). Theterm “yield” is art-recognized and includes the efficiency of theconversion of the carbon source into the product (i.e., Vitamin C). Thisis generally written as, for example, kg product per kg carbon source.By “increasing the yield and/or production/productivity” of the compoundit is meant that the quantity of recovered molecules, or of usefulrecovered molecules of that compound in a given amount of culture over agiven amount of time is increased. The terms “biosynthesis” or a“biosynthetic pathway” are art-recognized and include the synthesis of acompound, preferably an organic compound, by a cell from intermediatecompounds in what may be a multistep and highly regulated process. Thelanguage “metabolism” is art-recognized and includes the totality of thebiochemical reactions that take place in an organism. The metabolism ofa particular compound, then, (e.g., the metabolism of an amino acid suchas glycine) comprises the overall biosynthetic, modification, anddegradation pathways in the cell related to this compound. The language“transport” or “import” is art-recognized and includes the facilitatedmovement of one or more molecules across a cellular membrane throughwhich the molecule would otherwise either be unable to pass or be passedinefficiently.

Vitamin C as used herein may be any chemical form of L-ascorbic acidfound in aqueous solutions, such as for instance undissociated, in itsfree acid form or dissociated as an anion. The solubilized salt form ofL-ascorbic acid may be characterized as the anion in the presence of anykind of cations usually found in fermentation supernatants, such as forinstance potassium, sodium, ammonium, or calcium. Also included may beisolated crystals of the free acid form of L-ascorbic acid. On the otherhand, isolated crystals of a salt form of L-ascorbic acid are called bytheir corresponding salt name, i.e. sodium ascorbate, potassiumascorbate, calcium ascorbate and the like.

In one preferred embodiment, the present invention is related to aprocess for the production of Vitamin C wherein a nucleotide accordingto the invention as described above is inactivated in a suitablemicroorganism by methods described above, the recombinant microorganismis cultured under conditions that allow the production of Vitamin C inhigh productivity, yield, and/or efficiency, the produced fermentationproduct is isolated from the culture medium and optionally furtherpurified.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents and published patent applications, citedthroughout this application are hereby incorporated by reference.

EXAMPLES Example 1 Preparation of Chromosomal DNA and Amplification ofDNA Fragment by PCR

Chromosomal DNA of Gluconobacter oxydans IFO 3293 was prepared from thecells cultivated at 30° C. for 1 day in mannitol broth (MB) liquidmedium consisting of 25 g/1 mannitol, 5 g/l of yeast extract (Difco),and 3 g/l of Bactopeptone (Difco) by the method described by Sambrook etal (1989) “Molecular Cloning: A Laboratory Manual/Second Edition”, ColdSpring Harbor Laboratory Press).

A DNA fragment was prepared by PCR with the chromosomal DNA preparedabove and a set of primers, Pf (SEQ ID NO:3) and Pr (SEQ ID NO:4). Forthe reaction, the Expand High Fidelity PCR kit (Roche Diagnostics) and10 ng of the chromosomal DNA was used in total volume of 100 μLaccording to the supplier's instruction to have the PCR productcontaining SMS 27 DNA sequence (SEQ ID NO:1). The PCR product wasrecovered from the reaction and its correct sequence confirmed.

Example 2 Disruption of the SMS 27 Gene in G. oxydans IFO 3293

In order to construct a knockout mutant of the SMS 27 gene,Long-Flanking Homology (LFH) PCR was used to construct a cleandeletion-insertion mutation. Firstly, the upstream and downstreamregions flanking the SMS 27 gene were amplified by PCR using therespective primer pairs SMS 27LFH+1 (SEQ ID NO:5)/SMS 27 KmLFH-1 (SEQ IDNO:6) and SMS 27 KmLFH+1 (SEQ ID NO:7)/SMS 27LFH-1 (SEQ ID NO:8). G.oxydans IFO 3293 genomic DNA was used as a template and the reactionconditions consisted of 30 cycles of denaturation at 95° C. for 1 min,annealing at 50° C. for 1 min and extension at 72° C. for 1.5 min. Inboth cases, the Herculase DNA polymerase (Stratagene) was used tominimize PCR-generated errors. The kanamycin-resistance cassette wasamplified using pUC4K plasmid DNA (Amersham Bioscience, accession No.X06404) as a template and primer pair SMS 27 Km-1 (SEQ ID NO:9)/SMS 27Km+1 (SEQ ID NO:10) to generate a 1.3-kb fragment. The reactionconditions were as above. The three products were gel-purified, mixedand used in the second round PCR reaction with the flanking primers SMS27LFH+1/SMS 27LFH-1 to generate a product of 2.6-kb. The reactionconditions for the second round reaction consisted of 94° C., 2 min,then 10 cycles of [94° C., 30 sec, 63° C., 30 sec, 68° C., 6 min],followed by 20 cycles of [94° C., 30 sec, 63° C., 30 sec, 68° C., 6 minwith an additional 20 sec per cycle] and a final extension at 68° C. for10 min.

The PCR product was cloned into the pGEM-TEasy vector (Promega) to giveplasmid pGEM-SMS 27. This plasmid was transformed into competent G.oxydans IFO 3293 cells selecting transformants on MB agar mediumcontaining kanamycin to a final concentration of 50 μg ml⁻¹, resultingin mutant strain G. oxydans IFO 3293-SMS 27::Km. PCR using the flankingprimers SMS 27LFH+1/SMS 27LFH-1 was used to confirm that the SMS 27::Kmmutation had integrated via a double crossover.

Example 3 Production of Vitamin C from D-Sorbitol Using Resting Cells

Cells of G. oxydans IFO 3293 and G. oxydans IFO 3293-SMS 27::Km wereplated firstly on MB medium for three days. Then cells were scraped offthese plates and spread onto No. 3BD medium containing 7% sorbitol andgrown for 3 days at 30° C. These cells were used in resting-cellreactions with 2% sorbitol as substrate and a cell density of OD₆₀₀=10.

After 20 h incubation time, G. oxydans IFO 3293 produced 30 mg/l ofVitamin C from D-sorbitol. In comparison, strain G. oxydans IFO 3293-SMS27::Km produced more than 350 mg/l of Vitamin C.

Example 4 Production of 2-KGA from D-Sorbitol Using Resting Cells

In accordance with the experimental setup of Example 3, cells of strainsG. oxydans IFO 3293 and G. oxydans IFO 3293-SMS 27::Km were tested fortheir ability to produce 2-KGA from D-sorbitol.

After 20 h incubation time, G. oxydans IFO 3293 produced 700 mg/l of2-KGA from D-sorbitol. In comparison, strain G. oxydans IFO 3293-SMS27::Km produced more than 7 g/l of 2-KGA.

Example 5 Presence of the SMS 27 Gene and Equivalents in Other Organisms

The presence of SEQ ID NO:1 and/or equivalents showingsimilarity/identity to SEQ ID NO:1 in other organisms than the onesdisclosed herein before, e.g. organisms as mentioned in Table 1, may bedetermined by a simple DNA hybridization experiment.

Strains of Acetobacter aceti subsp. xylinum IFO 13693 and IFO 13773 aregrown at 27° C. for 3 days on No. 350 medium containing 5 g/lBactopeptone (Difco), 5 g/l yeast extract (Difco), 5 g/l glucose, 5 g/1mannitol, 1 μl MgSO₄.7H₂O, 5 ml/l ethanol, and 15 g/l agar. All otherAcetobacter, Gluconacetobacter and all Gluconobacter strains are grownat 27° C. for 3 days on mannitol broth (MB) agar medium containing 25g/1 mannitol, 5 g/l yeast extract (Difco), 3 g/l Bactopeptone (Difco),and 18 g/l agar (Difco). E. coli K-12 is grown on Luria Broth agarmedium. The other strains/cells are grown on medium recommended by thesuppliers or according to methods known in the art. Genomic DNA isextracted as described by e.g. Sambrook et al., 1989, “MolecularCloning: A Laboratory Manual/Second Edition”, Cold Spring HarborLaboratory Press) from a suitable organism as e.g. mentioned in Table 1.

Genomic DNA preparations are digested with restriction enzymes such asEcoRI or HindIII, and 1 μg of the DNA fragments are separated by agarosegel electrophoresis (1% agarose). The gel is treated with 0.25 N HCl for15 min and then 0.5 N NaOH for 30 min, and then blotted ontonitrocellulose or a nylon membrane with Vacuum Blotter Model 785(BIO-RAD Laboratories AG, Switzerland) according to the instruction ofthe supplier. The resulting blot is then brought into contact/hybridizedwith a solution wherein the probe, such as e.g. a DNA fragment with SEQID NO:1 sequence or a DNA fragment containing the part or whole of theSEQ ID NO:1 sequence to detect positive DNA fragment(s) from a testorganism. A DIG-labeled probe, e.g. SEQ ID NO:1, may be preparedaccording to Example 1 by using the PCR-DIG labeling kit (RocheDiagnostics) and a set of primers, SEQ ID NO:3 and SEQ ID NO:4 accordingto the supplier's protocol. A result of such a blot is depicted in Table1.

The hybridization may be performed under stringent or highly stringentconditions. A preferred, non-limiting example of such conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 1×SSC, 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 60° C. and even more preferablyat 65° C. Highly stringent conditions include, for example, 2 h to 4days incubation at 42° C. in a solution such as DigEasyHyb solution(Roche Diagnostics GmbH) with or without 100 μg/ml salmon sperm DNA, ora solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2%blocking reagent (Roche Diagnostics GmbH), followed by washing thefilters twice for 5 to 15 min in 2×SSC and 0.1% SDS at room temperatureand then washing twice for 15-30 min in 0.5×SSC and 0.1% SDS or 0.1×SSCand 0.1% SDS at 65-68° C. To detect DNA fragments with lower identity tothe probe DNA, final washing steps can be done at lower temperaturessuch as 50-65° C. and for shorter washing time such as 1-15 min.

The genes corresponding to the positive signals within the respectiveorganisms shown in Table 1 can be cloned by a PCR method well known inthe art using genomic DNA of such an organism together with a suitableprimer set, such as e.g. SEQ ID NO:3 and SEQ ID NO:4 under conditions asdescribed in Example 1 or as follows: 5 to 100 ng of genomic DNA is usedper reaction (total volume 50 μl). Expand High Fidelity PCR system(Roche Diagnostics) can be used with reaction conditions consisting of94° C. for 2 min; 30 cycles of (i) denaturation step at 94° C. for 15sec, (ii) annealing step at 60° C. for 30 sec, (iii) synthesis step at72° C. for 0.5 to 5 min depending to the target DNA length (1 min/1 kb);extension at 72° C. for 7 min. Alternatively, one can perform a PCR withdegenerate primers, which can be synthesized based on SEQ ID NO:2 oramino acid sequences as consensus sequences selected by aligning severalamino acid sequences obtained by a sequence search program such asBLASTP (or BLASTX when nucleotide sequence is used as a “querysequence”) to find proteins having a similarity to the protein of SEQ IDNO:2. For PCR using degenerate primers, temperature of the secondannealing step (see above) can be lowered to 55° C., or even to 50-45°C. A result of such an experiment is shown in Table 1.

Samples of the PCR reactions are separated by agarose gelelectrophoresis and the bands are visualized with a transilluminatorafter staining with e.g. ethidium bromide, isolated from the gel and thecorrect sequence is confirmed.

Consensus sequences mentioned above might be amino acid sequencesbelonging to certain categories of several protein domain/familydatabases such as PROSITE (database of protein families and domains),COGs (Cluster of Ortholog Groups), CDD (Conserved Domain Databases),pfam (large collection of multiple sequence alignments and hidden Markovmodels covering many common protein domains and families). Once one canselect certain protein with identical/similar function to the protein ofthis invention from proteins containing domain or family of suchdatabases, corresponding DNA encoding the protein can be amplified byPCR using the protein sequence or its nucleotide sequence when it isavailable in public databases.

Example 6 Disruption of the SMS 27 Gene and Equivalents in OtherOrganisms for Production of Vitamin C and/or 2-KGA

In order to improve Vitamin C production in a suitable microorganismwhich is capable to directly produce Vitamin C from a given substrate,the SMS 27 gene and equivalents as e.g. a PCR product obtained inExample 5, referred to hereafter as gene X, can be disrupted inaccordance to the SMS 27 gene in G. oxydans IFO 3293 (see Example 2) togenerate a knockout mutant carrying SMS 27 equivalent gene::Km. Suitablehost strains for generation of such knockout mutants may be selectedfrom e.g. Gluconobacter strains listed in Table 1, in particular e.g. G.oxydans IFO 3292, G. oxydans ATCC 621H, G. oxydans IFO 12528, G. oxydansIFO 3291, G. oxydans IFO 3255, G. oxydans IFO 3244, G. cerinus IFO 3266,G. frateurii IFO 3260, G. oxydans IFO 3287, Acetobacter aceti subsp.orleanus IFO 3259, Acetobacter aceti subsp. xylinum IFO 13693,Acetobacter aceti subsp. xylinum IFO 13773 and Acetobacter sp. ATCC15164, and their derivatives carrying genes involved in Vitamin Cproduction pathways and the adjacent regions where the SMS 27 gene orits equivalent might be located.

The knockout mutant such as, e.g. a knockout mutant G. oxydans IFO3292-SMS 27 equivalent gene::Km can be generated as follows: the PCRproduct obtained from G. oxydans IFO 3293 described in Example 5 iscloned in an E. coli vector pCR2.1-TOPO and used to transform E. coliTG1 to have a Ap^(r) transformant carrying pCR2.1-gene X. Then, Km^(r)cassette isolated from pUC-4K (Amersham Bioscience, accession No.X06404) is inserted into one of the restriction site of the target genewith ligase and the resulting ligation product is used to transform E.coli TG1 to have Apr Kmr transformant carrying pCR2.1-gene X::Km. ThepCR2.1-gene X::Km plasmid prepared from the transformant is digested bytwo restriction enzymes selected from the multi-cloning site of thevector part to isolate a DNA fragment containing gene X::Km. Theresulting DNA fragment is used to transform the host strain carrying theSMS 27 equivalent gene by electroporation to have the gene disruptantcarrying SMS 27 equivalent gene::Km.

Further modifications including genes involved in the conversion ofD-sorbitol, L-sorbose and/or L-sorbosone into Vitamin C within saidstrains may be generated to improve Vitamin C production within suchstrains.

Production of Vitamin C using the cells of the knockout mutant, e.g. G.oxydans IFO 3292-SMS 27 equivalent gene::Km, and the correspondingwild-type strain, e.g. G. oxydans IFO 3292, are performed according toExample 3.

Production of 2-KGA using the cells of the knockout mutant, e.g. G.oxydans IFO 3292-SMS 27 equivalent gene::Km, and the correspondingwild-type strain, e.g. G. oxydans IFO 3292, are performed according toExample 4.

In the resting cell reaction with 1% L-sorbosone as the substrate, themutant strain can produce at least more than 20% Vitamin C and at leastmore than 10% 2-KGA compared to the wild-type strain.

TABLE 1 Equivalents of the SMS 27 gene in other organisms. Sig- Sig-Sig- Strain nal 1 nal 2 nal 3 G. oxydans IFO 3293 + + + G. oxydans DSM17078 + + + G. oxydans IFO 3292 + + + G. oxydans ATCC 621H + + + G.oxydans IFO 12528 + + + G. oxydans G 624 + − + G. oxydans T-100 + + + G.oxydans IFO 3291 + − + G. oxydans IFO 3255 + − + G. oxydans ATCC 9937 +− + G. oxydans IFO 3244 + − + G. cerinus IFO 3266 + − + G. frateurii IFO3260 + − + G. oxydans IFO 3287 + − + Acetobacter aceti subsp. orleanusIFO 3259 − − + Acetobacter aceti subsp. xylinum IFO 13693 − − +Acetobacter aceti subsp. xylinum IFO 13773 − − + Acetobacter sp. ATCC15164 − − + G. thailandicus NBRC 100600 + − + Gluconacetobacterliquefaciens ATCC 14835 + − + Gluconacetobacter polyoxogenes NBI 1028 −− + Gluconacetobacter diazotrophicus ATCC 49037 − − + Gluconacetobactereuropaeus DSM 6160 − − + Acetobacter aceti 1023 − − + Acetobacterpasteurianus NCI 1193 − − + E. coli − − − Saccharomyces cerevisiae − − −Aspergillus niger − − − Mouse − − − Signal 1: Detection of DNA on a blotwith genomic DNA of different strains and SEQ ID NO: 1 as labeled probe.Signal 2: Detection of DNA of different strains in a PCR reaction usingprimer pair SEQ ID NO: 3 and SEQ ID NO: 4. Signal 3: Detection of DNA ofdifferent strains in a PCR reaction using degenerate primers. For moreexplanation refer to the text.

1. A polynucleotide selected from the group consisting of: (a)polynucleotides encoding a polypeptide comprising the amino acidsequence according to SEQ ID NO:2; (b) polynucleotides comprising thenucleotide sequence according to SEQ ID NO:1; (c) polynucleotidescomprising a nucleotide sequence obtainable by nucleic acidamplification such as polymerase chain reaction, using genomic DNA froma microorganism as a template and a primer set according to SEQ ID NO:3and SEQ ID NO:4; (d) polynucleotides comprising a nucleotide sequenceencoding a fragment or derivative of a polypeptide encoded by apolynucleotide of any of (a) to (c) wherein in said derivative one ormore amino acid residues are conservatively substituted compared to saidpolypeptide, and said fragment or derivative has the activity of atranscriptional regulator, preferably a repressor of L-sorbosedehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH); (e)polynucleotides the complementary strand of which hybridizes understringent conditions to a polynucleotide as defined in any one of (a) to(d) and which encode a transcriptional regulator, preferably a repressorof L-sorbose dehydrogenase (SDH) and/or L-sorbosone dehydrogenase(SNDH); and (f) polynucleotides which are at least 60%, such as 70, 85,90 or 95% identical to a polynucleotide as defined in any one of (a) to(d) and which encode a transcriptional regulator, preferably a repressorof L-sorbose dehydrogenase (SDH) and/or L-sorbosone dehydrogenase(SNDH); or the complementary strand of such a polynucleotide.
 2. Avector containing the polynucleotide according to claim
 1. 3. The vectorof claim 2 in which the polynucleotide is operatively linked toexpression control sequences allowing the expression in prokaryotic oreukaryotic host cells.
 4. A microorganism genetically engineered with apolynucleotide according to claim
 1. 5. A microorganism according toclaim 4 capable of directly producing Vitamin C from D-sorbitol inquantities of 300 mg/l or more when measured in a resting cell methodafter an incubation period of 20 hours.
 6. A microorganism according toclaim 5 capable of directly producing Vitamin C from L-sorbose inquantities of 800 mg/l or more.
 7. A polypeptide encoded by apolynucleotide according to claim
 1. 8. Process for producing cellscapable of expressing a polypeptide according to claim 7, comprising thestep of genetically engineering cells with the vector.
 9. Use of adisrupted polynucleotide according to claim 1 for the production ofVitamin C and/or 2-KGA.
 10. A microorganism according to claim 4 or amicroorganism containing an endogenous gene comprising a polynucleotide,said microorganism being genetically altered in such a way that it leadsto an improved yield and/or efficiency of production of Vitamin C and/or2-KGA produced by said microorganism.
 11. A microorganism according toclaim 10 producing a polypeptide with decreased or abolished activity ofa transcriptional regulator, preferably a repressor of L-sorbosedehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH) gene.
 12. Amicroorganism according to claim 4 wherein the polynucleotide isdisrupted.
 13. A microorganism according to claim 4 selected from thegroup consisting of Pseudomonas, Pantoea, Escherichia,Ketogulonicigenium and acetic acid bacteria like e.g., Gluconobacter,Acetobacter or Gluconacetobacter, preferably Acetobacter sp.,Acetobacter aceti, Gluconobacter frateurii, Gluconobacter cerinus,Gluconobacter thailandicus, Gluconobacter oxydans, preferablyGluconobacter oxydans, more preferably Gluconobacter oxydans IFO 3293.14. Process for the production of an disrupted endogenoustranscriptional regulator gene, preferably a repressor of L-sorbosedehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH) gene in amicroorganism, said microorganism comprising a polynucleotide accordingto claim 1, said process comprising the step of altering saidpolynucleotide in such a way that it leads to an improved yield and/orefficiency of production of Vitamin C and/or 2-KGA produced by saidmicroorganism.
 15. Process for the production of a microorganism capableof producing Vitamin C and/or 2-KGA, comprising the step of alteringsaid microorganism so that the microorganism produces a polypeptide withreduced or abolished activity of a transcriptional regulator, preferablya repressor of L-sorbose dehydrogenase (SDH) and/or L-sorbosonedehydrogenase (SNDH) gene leading to an improved yield and/or efficiencyof production of Vitamin C and/or 2-KGA produced by said microorganism.16. Process for the production of a microorganism containing anendogenous gene comprising a polynucleotide according to claim 1,comprising the step of altering said microorganism so that theendogenous gene is underexpressed or disrupted, leading to an improvedyield and/or efficiency of production of Vitamin C and/or 2-KGA producedby said microorganism.
 17. Process according to claim 15 the productionof a microorganism.
 18. Microorganism obtainable by a process accordingto claim
 14. 19. Process for the production of Vitamin C and/or 2-KGAwith a microorganism according to claim 10 wherein said microorganism isincubated/cultivated in a aqueous medium under conditions that allow thedirect production of Vitamin C and/or 2-KGA from D-sorbitol or L-sorboseand wherein optionally Vitamin C and/or 2-KGA is isolated as thefermentation product.